Ginger as an anticolorectal cancer spice: A systematic review of in vitro to clinical evidence

Abstract Ginger and its derivatives have been shown to be effective in the prevention and treatment of cancer. We undertook a systematic review to answer the question of whether ginger has a role in modifying the biomarkers of cancer in cell culture conditions and on colorectal cancer in randomized clinical trials. We performed a comprehensive search of the literature from Scopus, Embase, Web of Science, PubMed, Cochrane central register of controlled trials, and Cochrane database of systematic reviews. At first, all 12 papers studied the effect of ginger or its derivatives on cell culture conditions. The results of cell culture studies show that ginger has a powerful role in inducing apoptosis. In the second part, five studies of clinical trials were analyzed. By analyzing antitumor markers of clinical trials, ginger increased some anticancer markers but performed poorly in inducing some anticancer markers. This systematic review showed that the consumption of ginger extract has the potential to prevent and treat colorectal cancer but this ability is weak.


| INTRODUC TI ON
Colorectal cancer (CRC) is a common malignant tumor with high morbidity and mortality worldwide. The most crucial cause of colon cancer can be considered aging and unhealthy lifestyle as well as genetic predisposition. Among unhealthy lifestyle factors, there is growing evidence about the roles of dietary components such as red meats, processed meats, and alcohol in the occurrence and development of CRC (Bent & Ko, 2004). On the other hand, some nutraceuticals in specific foods have benefits as well as few side effects in arresting cancer cells.
Ginger (Zingiber officinale) has been used worldwide as a spice, condiment, and medicinal remedy in many countries. Ginger and its active compounds, such as 6-gingerol and 6-shogaol, have shown beneficial biological effects including hepatoprotective, anti-inflammation, antioxidation, and anticancer activities Samadi et al., 2022). In many in vitro studies, it has been reported that ginger has a high ability to induce apoptosis in a variety of cancer cells (Arablou et al., 2014;Shidfar et al., 2015). One research has shown that ginger components such as 6-and 10-gingerols have a beneficial role in the treatment of cervical cancer (Zhang et al., 2017). 6-gingerol has inhibited cell proliferation, induced apoptosis, and blocked G1 cell-cycle arrest in human colorectal cancer cells (Lee et al., 2008). In addition, it can induce apoptosis in human colorectal carcinoma cells through the activation of caspases (cysteine-aspartic proteases, cysteine aspartate, or cysteine-dependent aspartate-directed proteases) and the production of reactive oxygen species (Lee et al., 2008). Several studies have shown that gingerol modulates a variety of cell signaling pathways linked to cancer, including nuclear factors (NF-κB), signal transducer and activator of transcription 3 (STAT3), activator protein-1 (AP-1), βcatenin, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), mitogen-activated protein kinases (MAPK), and pro-inflammatory mediators such as tumor necrosis factor (TNFα) and cyclooxygenase-2 (COX-2) (Jemal et al., 2009;Ling et al., 2010). In some studies, it has been also shown that the anticancer activity of ginger extract is significantly higher than curcumin in brain cancer cells .
Since the cancer cell line models are a very useful diagnostic tool for the diagnosis of cancer at the starting point, most studies investigated the anticancer role of ginger in cell culture (Mirabelli et al., 2019). Questions in this study are as follows: Does ginger have a role in modifying the biomarkers of cancer in cell culture conditions? Does ginger have a role in modifying the biomarkers in colorectal cancer in clinical trials? At the end of this study, we compared the changes in cancer biomarkers in both conditions.

| Study design
This systematic review was performed by the preferred reporting items of systematic reviews and meta-analyses (PRISMA) statement (Liberati et al., 2009). This systematic review was registered in the International Prospective Register of Systematic Reviews (PROSPERO) in 2022 (ID:CRD42022369388). Briefly, the aim is to identify the anticancer effects of ginger in cancer cell line models and clinical trials.

| Search strategy
We systematically searched the Scopus, Embase, Web of Science, PubMed, Cochrane central register of controlled trials (CCTR), and Cochrane database of systematic reviews (CDSR) databases.
Keywords for search in these databases were included: The study was complemented by a search for these keywords in the databases mentioned above for eligible articles and email correspondences with authors for additional data where relevant.
We included all cell cultures and randomized clinical trials that had studied the anticancer effect of ginger in human or human cancer cells.

| Study selection
Study selection started with the removal of duplicates, followed by titles and abstracts to answer our question screening by two independent reviewers. To avoid bias, they were blinded to the names, qualifications, or institutional affiliations of the study authors. The full text of studies meeting inclusion criteria was retrieved and screened to determine eligibility by two reviewers. Following the assessment of methodological quality, two researchers extracted data using a purpose-designed data extraction form and independently summarized what they considered to be the most important results from each study. These summaries were compared, and then any differences of opinion were resolved by discussion and consultation with a third reviewer. Any further calculations on study data considered necessary were conducted by the first reviewer and checked by the second reviewer. If the two authors failed to reach a consensus, the third author was involved in making a final decision.

| Eligibility criteria
We included studies in this review if they were conducted on the anticancer effects of ginger at the cell culture condition and randomized clinical trials (RCT) that were published in the English language. Also, the studies included in this paper are clinical trials involving human participants with normal and increased risk of colorectal cancer.
Exclusion criteria were as follows: articles that did not interfere with Ginger; articles that do not investigate cancer in cell culture and do not involve human participants with colorectal cancer; and articles that do not investigate the role of ginger in preventing and treating cancer.

| Methodological quality assessment
The Cochrane risk-of-bias tool for randomized trials was used to assess the quality of the studies included in this systematic review. This tool consists of five bias domains, including selection bias (random sequence generation and allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete data outcome), and reporting bias (selective outcome reporting). We categorized clinical trial studies as Yes (low risk of bias), No (high risk of bias), or Unclear for each domain. Finally, the overall quality of the studies was categorized into weak, fair, or good, if <3, 3, or ≥4 domains were rated as low risk, respectively (Higgins et al., 2019;Raeisi-Dehkordi et al., 2019).

| Data extraction
In the present study, we analyzed several factors to evaluate the effect of ginger in preventing or treating cancer. One of the most important and key pathways to understanding the anticancer role of ginger is to study apoptosis. Therefore, we analyzed caspases, B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), and p21 to investigate the role of ginger in inducing apoptosis. Matrix metalloproteinases (MMPs) were analyzed to investigate the role of ginger in the proliferative factors. Also, to investigate the anti-inflammatory effect of ginger, we analyzed the role of ginger in reducing inflammatory factors and enzymes involved in the inflammatory process such as cyclooxygenases (COXs), 5-lipoxygenase (5-LOX), 12-LOX, 15-LOX-2, prostaglandin E2 (PGE2), and 15-hydroxyeicosatetraenoic acid (15-HETE).

| RE SULTS
We initially identified 1298 nonduplicate articles through a systematic search. After appraising the title/abstract of articles, 1233 articles were excluded due to not meeting inclusion criteria. Of the 65 remained articles, finally, 17 full-text articles were selected that could gain inclusion criteria ( Figure 1). Twelve papers have evaluated the effect of ginger or its derivatives on cell culture conditions, and five papers have evaluated the effect of ginger and its derivatives on colorectal cancer biomarkers in clinical trials.

| Biomarker analyzed on in vitro study
Here are six articles that studied molecular biomarkers (Akimoto et al., 2015;Choudhury et al., 2010;Lee et al., 2008;Miyoshi et al., 2003;Saha et al., 2014), that the molecular factors of Bax and Bcl2 studied widely. Studies showed that Bax is increased and induced the opening of the mitochondrial voltage-dependent anion channel, so the death of the cell is guaranteed (Mignard et al., 2014). The Bcl-2 family of regulator proteins regulated apoptosis, by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosis. In all of these studies, it has been shown that the amount of Bcl-2 in the treated cells with ginger has decreased, and conversely, Bax has increased.
Seven articles studied the role of proliferative factors and MMPs (Lee et al., 2008;Ling et al., 2010;. MMPs have been implicated as possible mediators of invasion and metastasis in some cancers . Five studies have studied the anti-inflammatory effects and these papers have shown that treating cells with ginger reduced inflammation (Ishiguro et al., 2007;Ling et al., 2010;Miyoshi et al., 2003;Romero et al., 2018;Saha et al., 2014). Also, four papers have proven that ginger has increased the activity of caspases (Choudhury et al., 2010;Ishiguro et al., 2007;Miyoshi et al., 2003;. All in vitro studies have shown that the treatment of cells with ginger stops their cell growth and causes cell death ( Table 1).

| Biomarker analyzed on randomized clinical trials
Five clinical trials have studied the effect of ginger and its derivatives on colorectal cancer. Table 2 shows the quality of the studies based on the Cochrane risk-of-bias tool. Of these ( Table 3), four have been conducted in the United States (Citronberg et al., 2013;Jiang et al., 2013;Zick et al., 2011;Zick et al., 2015). Another article from Thailand studied patients with colorectal cancer (Danwilai et al., 2017). trointestinal symptoms in five subjects (35.7%) and headache in one subject (7.14; discontinue) with ginger consumption among subjects at normal risk for colorectal cancer (Zick et al., 2011). In another study, the serum levels of alanine aminotransferase were significantly increased in two subjects (from 35 to 65 and 31 to 42 U/L) (Danwilai et al., 2017). No significant adverse events related to ginger extract were observed in both studies.

| DISCUSS ION
Apoptosis is programmed cell death that provides instructions and conditions for cancer cell death (Wlodkowic et al., 2011). Caspases are a family of protease enzymes and play essential roles in programmed cell death (Galluzzi et al., 2016). In vitro studies have shown that treatment with ginger stops cancer cell growth and causes cell death through the activation of Bax. This protein increases and induces the opening of the mitochondrial voltage-dependent anion channel, so the death of the cell is guaranteed (Mignard et al., 2014).
The Bcl-2 is a family of proteins regulating apoptosis, by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosis. The in vitro studies have shown that the amount of Bcl-2 in the treated cells with ginger has decreased, while Bax protein has increased. In addition, several in vitro studies examined the role of proliferative factors and MMPs (Choudhury et al., 2010;Ishiguro et al., 2007;Ling et al., 2010;Rhode et al., 2007).

MMPs have been implicated as possible mediators of invasion and
metastasis in some cancers . In vitro studies showed that ginger has a powerful role in inhibiting COXs. These enzymes are responsible for the formation of thromboxane and prostaglandins from arachidonic acid (Ogunwobi et al., 2012). Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin exert their effects through the inhibition of COX. One of the primary effectors of COX-dependent mechanisms in carcinogenesis is likely to be prostaglandins in particular PGE2. This prostaglandin increased cellular proliferation, migration, and invasiveness, promotes angiogenesis, induces resistance to apoptosis, and modulates cellular and humoral immunity (Greenhough et al., 2009). Aspirin's most well-characterized pharmacologic activity is the permanent modification of the COX enzymes (Patrono et al., 2005;Sankaranarayanan et al., 2020). There is also a non-COX-related pathway that might mediate aspirin's anticancer effects by suppressing nuclear factor kappa B (NF-κB) expression (Fu et al., 2019).
To identify the more precise anticancer mechanisms of ginger, image analysis (Citronberg et al., 2013). The results were contrary to the results of in vitro studies. They showed that Bax increased in recipients of ginger, but results not show a change in Bcl2 and p21. However, p21 is a potent inducer of differentiation in intestinal colonocytes. It has been reported that the expression of Bax in the early stages of tumorigenesis in the colon is reduced, and abnormalities in p21 expression have been linked to carcinogenesis (Pryczynicz et al., 2014;Shukla & Singh, 2007). It should be noted that the studies were conducted on people at risk of colorectal cancer and not on people with colorectal cancer (Citronberg et al., 2013;Jiang et al., 2013;Zick et al., 2011;Zick et al., 2015).
Somehow, these individuals do not have abnormal biomarkers and their biomarkers are in normal condition, in other words, if Bcl2 and p21 have not increased, also we should not expect molecular markers to change in these individuals. A remarkable point in these studies is that the amount of proliferative factors, such as hTERT and MIB-1 (Ki-67), has been significantly reduced. But the interesting tip is that ginger has been able to reduce hTERT and MIB-1 (Ki-67) in these individuals. A decrease in hTERT expression is consistent with previous reports, which found that ginger inhibited hTERT and c-Myc expression in human lung cancer cells (Tuntiwechapikul et al., 2010). Oncogenes activate hTERT, while tumor suppressor p53 inhibits cancerous cell growth (Kyo et al., 2008). The MIB-1 (Ki-67) protein is a cellular marker for the proliferation of cells. It is strictly associated with cell proliferation (Cuylen et al., 2016). However, factor MIB-1 (Ki-67) decreased (Citronberg et al., 2013). One of the most important deficiencies in five clinical trials was the lack of clarification of the quantities of ginger derivatives in the blood. In other words, ginger derivatives have not been reported in the serum. The key point is that logically ginger may have the ability to activate or inhibit some of the pathways controlling cancer, and not all of them.
The main strength of this study was that the effect of ginger on cancer was compared both in cell culture conditions and in experimental conditions. In addition, this study was that each cancer marker was carefully analyzed in both conditions. One of the limitations of this study was that the number of clinical trial studies was not large and there is a need to investigate the effect of ginger on colorectal cancer in different places. Another limitation of this study was that the components of ginger in the serum of people were not investigated. If ginger compounds are measured in the serum of people, the effect of digestion and absorption on its consumption will be determined. Also, one of the significant drawbacks of the clinical trials studied was that none of these trials measured ginger derivatives in blood serum. Somehow, ginger and its derivatives are probably metabolized in the body so its effects are contradictory.

| CON CLUS ION
In vitro studies consistently showed the beneficial effect of ginger on the treatment and prevention of cancer cells in cell culture models, but the results of clinical trials do not justify these results. The results obtained from clinical trials have inconsistent. In part of clinical trial studies, the anticancer effect of ginger has been proven, and in the other part, this hypothesis has been rejected. This indicated that ginger may play a role in the prevention and treatment of cancer, but this is not a dominant role.